Ukraine raised concerns about a hold on military aid on the same day as Donald Trump’s infamous phone call with its president, a Pentagon official told the impeachment inquiry on Wednesday.

The evidence from Laura Cooper, a deputy assistant secretary of defence, knocks down a key pillar of the White House’s defence: that Ukraine was unaware of the suspension of nearly $400m in security assistance until a much later date.

Cooper told the House intelligence committee that her staff received an email on 25 July from the state department saying that Ukraine’s embassy and the House foreign affairs committee were asking about military aid.

Sondland's bombshell testimony blows holes in Trump's Ukraine defence Read more

“On July 25, a member of my staff got a question from a Ukraine embassy contact asking what was going on with Ukraine security assistance,” Cooper testified. “Because at that time we did not know what the guidance was … I was informed that the staff member told the Ukrainian official that we were moving forward but recommended that the Ukraine embassy check in with state [department]”.

Asked later if the question referred specifically to the suspended aid, Cooper confirmed: “There was an awareness of that and there was an expression of concern.”

On the morning of 25 July, Trump spoke by phone with the Ukrainian president, Volodymyr Zelenskiy, asking for a “favour” and seeking investigations into a gas company linked to the former vice-president Joe Biden’s family and a debunked conspiracy theory about Ukrainian meddling in the 2016 election.

A day earlier, the special counsel Robert Mueller had testified to Congress about the findings of his report on Russian election interference, after which Trump tried to claim vindication. On 26 July, Trump was overheard talking about “investigations” on a phone call to the US ambassador to the EU, Gordon Sondland, who was meeting Ukrainian officials in Kiev.

Play Video 3:15 Trump impeachment inquiry: key moments from Sondland's explosive testimony – video

Cooper’s evidence, of which she was not aware when she testified behind closed doors, is significant because Republicans have claimed that Ukraine was only aware of the hold on military aid for two weeks before it was lifted on 11 September. It suggests that the Ukrainian president might have felt pressure to announce investigations from the day of the phone call onward.

The Democratic congressman Eric Swalwell told the witness her testimony “destroys two of the pillars of the president’s defence” – that Ukraine did not know about the aid freeze and that Trump is a champion of anti-corruption efforts.

Cooper also testified that she had never discussed a hold on security assistance for Ukraine with Trump himself and never heard from him directly on the matter.

As the hearing went on with Cooper and David Hale, under secretary of state for political affairs, Trump remained defiant. “If this were a prizefight, they’d stop it!” he tweeted.